Method of isolating a spore-forming mosquito larvicide

ABSTRACT

A method of controlling mosquito larvae, by using a spore-forming bacillus ONR-60A obtained from screening clonal isolates from soil samples of known mosquito larval breeding sites. A larvicide comprising the bacillus and a carrier is formulated as a buoyant colloidal suspension which stabilizes just under the surface of the water to concentrate in the feeding zone of many varieties of mosquito larvae.

This application is a division of patent application Ser. No. 888,083filed Mar. 20, 1978, now U.S. Pat. No. 4,166,112, issued Aug. 28, 1979.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of mosquito larval control. Morespecifically, the invention relates to larval control by use of a novelspore-forming bacillus and a unique carrier for dispersal of thebacillus. In particular, the invention relates to a unique strain ofspore-forming bacillus belonging to the species Bacillus thuringiensisand a unique buoyant colloidal suspension by which the bacillus iscarried and dispersed into known habitats of mosquito larvae.

2. Description of the Prior Art

Two major approaches have evolved in the development of effective agentsfor larval control. One approach is the use of chemical compounds ofvarying toxicity. The other approach is the development and use ofbiological microorganisms with high larvicidal activity.

The major shortcoming of the first approach is that new compounds ofincreased toxicity must be developed or dosage levels of standardcompounds increased to counter elevated, adaptive resistance by insectpopulations to standard insecticides. An unfortunate byproduct of thisapproach to date is increased toxicity to other lifeforms within theregion of application.

Biological microorganisms to date demonstrate a lower or negligibledevelopment of resistance in host vectors. Such biologicalmicroorganisms may be developed offering improved lethality against hostvectors, improved specificity against particular host vectors, andbetter methods of application without correspondingly lethal effectsupon other lifeforms within the region of application.

Technology in the development of bacterial entomotoxins to controlmosquito larvae is well known. Some of the entomotoxins employed havebeen Bacillus sphaericus Neide, Bacillus sphaericus var fusiformis, anda bicrystalliferous strain of Bacillus thuringiensis. In particular,selected strains of Bacillus thuringiensis have been used in Europe andAmerica as major components of microbial insecticides having a toxiceffect on general agricultural and forest insect pests. (A. Krieg:Bacillus thuringiensis, Berliner, Berlin, 1961.) To date, however, useof Bacillus thuringiensis against insects directly harmful to humanswithout concomitant adverse effects has not been employed. The presentinvention is in part a unique strain of Bacillus thuringiensis,hereinafter referred to as ONR-60A, having high, long-lasting,selectively toxic effects on mosquito larvae with no apparent adverseeffects on other lifeforms.

Information gathered from all the above studies indicates that severalrequirements must be met by any microbial entomotoxin before it can besafely and practically used in the field. These requirements are: (1)effective larval control, e.g., effective dosage levels having 95%lethality when employed (ED₉₅), should require less than 10⁶ cells/cm²of larval pond surface area; (2) the toxin should be formulated as abuoyant colloidal suspension such that a toxic concentration willstabilize just below the surface of the larval habitat; (3) larvicidalactivity should be retained under conditions of heat and exposure toultraviolet radiation; and (4) the environmental impact following theuse of a selected entomotoxic formulation must not be damaging to adesired ecological balance or otherwise adversely affect other lifeformswithin the zone of entomotoxin application.

Regarding criteria (2) listed above, the natural breeding habitat ormosquito larvae is in ponds, lakes, streams, marshes and the like in theshallow littoral zone. Most mosquito larvae must surface for oxygen andas a consequence feed in the region just below the water's surface.Larvicides employed as oil films asphyxiate the larvae as they surfaceto breath but have the disadvantage that the oil slick is deletorious tomuch other flora and fauna as well. Further, field studies indicatelarvae avoid areas having such oil slicks.

Dust layer and oil film insecticides designed to float on the surface ofthe water are strongly affected by wind currents, water currents and thelike and have serious problems in application and maintenance. Typicalof such problems is the drifting and accumulation of such larvicidesupon land areas during application or the formation of scum layers onthe surface of the water that subsequently concentrate upon land areas.

Larvicidal formulations having non-buoyant characteristics aredisadvantageous in that they are largely ineffective if dispersed inwater having greater depth than the feeding zone of the larvae.Effectiveness is reduced even more where the non-buoyant formulation isa contact insecticide.

When microbial entomotoxins are employed, the specificity or range ofthe toxic effect of the particular microorganism is extremely importantsince it is this characteristic that principally determines its utilityand the extent of its use. For example, within the species Bacilluscereus, some variant strains have pathogenic effects limited to a narrowrange of insects while other strains have no observed toxic effects atall even though they have essentially identical morphologicalcharacteristics. (Ibuki, et al U.S. Pat. No. 3,651,215). As mentionedsupra, Bacillus thuringiensis another species of microorganism havinggreat variation of effects by strains within the species, has had littleuse to date as an entomotoxin against insects directly harmful tohumans. Although some strains have been isolated that are effectiveagainst mosquito larvae, they have too narrow a range to be of greatutility.

Table I illustrates the wide variation of larvicidal activity, ornon-activity, of many strains of Bacillus thuringiensis and clearlypoints out that larvicidal activity is not associated with any singleserotype and that the only real characterization or measure of utilityand novelty of a microbial entomotoxin is its larvicidal activity.Persons skilled in the art of working with such entomotoxins canroutinely cause trivial mutations of a particular entomotoxic agenthaving no significantly different larvicidal activity, but having somemorphological characteristics changed by the trivial mutation. Thepresent invention contemplates such trivial mutations.

                  TABLE I                                                         ______________________________________                                        Summary of screening of test culture of                                       Bacillus thuringiensis (BA068) for larvicidal                                 activity against Culex tarsalis (K.L.) 1st                                    instar test larvae.                                                           ______________________________________                                        Isolates Demonstrating Larvicidal Activity                                     Original Designation                                                                          Serotype    Code Number                                      ______________________________________                                        kurstaki         H3a,3b*     (2536-9693TW)                                    kurstaki          "          (2819-9763F)                                     aizawai          H7*         (1850-9762C)                                     tolworthi        H9*         (NPI-460)                                        sotto            H4a,4b      (NPI-180-5-2)                                    thuringiensis    H1          (NPI-186-104)                                    thuringiensis     "          (NPI-185-104)                                    thuringiensis     "          (NPI-201-113)                                    thuringiensis     "          (NPI-197-105)                                    thuringiensis     "          (NPI-198-105)                                    thuringiensis     "          (NPI-199-105)                                    sotto            H4a,4b      (NPI-194-101)                                    ______________________________________                                        Isolates Demonstrating No Larvicidal Activity                                 Original         Serotype    Code Number                                      ______________________________________                                        kurstaki         H3a,3b*     (720619A)                                        B. thuringiensis var.?                                                                         H5a,5b      (730130-1)                                       thuringiensis    H1*         (1840-182C)                                      finitimus        H2*         (NPI-451)                                        subtoxious       H6*         (NPI-456)                                        entomocidus      H6*         (NPI-457)                                        aizawai          H7*         (NPI-458)                                        morrisoni        H8*         (NPI-459)                                        darmastadiensis  H10*        (NPI-461)                                        ______________________________________                                         *Cultures marked with an asterisk have had recent confirmation. The           remainder are based on information available before 1963, and therefore,      there is a possibility those classifications are not accurate.           

SUMMARY OF THE INVENTION

The present invention may be briefly summarized as a method ofcontrolling mosquito larvae by using a spore-forming bacillus ONR-60Aobtained from screening clonal isolates from soil samples of knownmosquito larval breeding sites. The larvicide comprising the bacillusand a carrier is formulated as a buoyant colloidal suspension whichstabilizes just under the surface of the water to concentrate in thefeeding zone of mosquito larvae.

A primary object of the present invention is the development of a uniquemicrobial entomotoxin and a carrier having high, long-lasting and veryselective toxic effect on mosquito and mosquito-like larvae withoutadversely affecting the environment within the zone of application.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present inventor, having studied mosquito breeding habitats inIsrael discovered unique null zones of mosquito larvae activity withinregions of normally high mosquito and mosquito larvae activity, analyzeda number of soil samples from the null zones to determine the cause ofsuch unusually high larvicidal activity. The present inventor devised anempirical method for locating and isolating microorganisms havingunique, high larvicidal activity and in the instant case succeeded inisolating the microorganism herein referred to as ONR-60A. He furtherdevised a unique carrier for dispersal of the new larvicide.

A sample of the microorganism has been deposited with the InternationalCulture Depository, Columbus, Ohio, 43210. In subsequent screening andanalysis by WHO, it has been identified as a unique strain of Bacillusthuringiensis and has been granted an accession number within thisdepository as WHO/CCBC 1897.

The spores of ONR-60A were successfully isolated and a pure biologicalstrain obtained in the following way. Known mosquito larvae habitatswere searched and both moist and dry soil samples were taken from nullzones within the habitat wherein larvicidal activity within the soil wasuncommonly high and the larvae population uncommonly low. A liquiddilution was then experimentally determined to provide easily separableclonal isolation of the microorganisms contained in the samples. In theinstant case, 30-100 clones/agar plate was chosen as a manageablenumber. The plates were then inoculated to obtain a sufficient number ofwell-separated and distinctive clonal isolates. Inoculated plates wereincubated for 48 hours at 30° C. and then held at room temperature,22°-26° C., for approximately 48 additional hours to obtain a visuallyevident, clearly-defined clonal morphology. Several examples of eachclonal isolate were then selected from each test soil sample forscreening. A manageable number of such cloned isolates were screened inthe ensuing larval test runs. In the instant case, the number ofisolates screened per larval test run was approximately 100- 150.

Screening for each clone was accomplished by using a row of five wells,each well having 4 ml of sterile liquid and containing two or three testlarvae. Each row was then challenged using one drop/well, 0.04 ml/well,of a nominal 10⁹ cells/ml test slurry obtained by scrubbing the agarsurface growth with a dally rod and 5 ml of a sterile diluent. The testslurry obtained was approximately 10⁹ cells/ml, thus resulting in aninitial larval screening challenge of approximately 10⁷ cells/ml. Anyclone which demonstrated over 80% mortality in 24-48 hours wasconsidered for further quantitative analysis.

Clonal isolates selected for additional screening were inoculated ontoboth a nutrient agar and a test agar and incubated as required to get avisually-evident, clearly-defined clonal morphology on each agarsurface. The agar surface growths were removed by scrubbng each with adally rod and 5 ml of a sterile diluent. One or two trays of first tosecond instar C. pipiens complex larvae, for example, were then used ina 1/3 test challenge dilution series of each of the growths on each agarsurface with 15-30 larvae/test dilution. Each tray contained 25 wellswith three larvae/well. The clonal isolate ONR-60A, observed todemonstrate larvicidal activity independent of the agar surface on whichit was grown, was then produced in quantities for field test purposes bymethods familiar to those in the art.

Tables II and III illustrate the toxicity of ONR-60A against variousmosquito larvae species. Unlike prior strains of Bacillus thuringiensishaving too narrow a range of toxic effect, as mentioned supra, ONR-60Apossesses utility in application against a broad range of mosquitolarvae species.

                  TABLE II                                                        ______________________________________                                        Relative Toxicity, estimated ED.sub.50, of ONR-60A                            Against Different Species of Mosquito Larvae                                                         Concentration                                          Species                (cells/ml)                                             ______________________________________                                        Anopheles sergentii    5 * 10.sup.5                                           Anopheles superpictus  2 * 10.sup.5 +                                         Anopheles hyrcanus complex                                                                           2 * 10.sup.5                                           Anopheles stephensi    2 * 10.sup.5                                           Uranotaenia unguiculata                                                                              3 * 10.sup.4                                           Culex univittatus      2 * 10.sup.4                                           Aedes aegypti          1 * 10.sup.4                                           Culex pusillus         7 * 10.sup.3                                           Culex pipiens (m)      6 * 10.sup.3                                           Culex pipiens complex  5 * 10.sup.3 +                                         Culex theileri         3 * 10.sup.3 +                                         Culex torrentium       3 * 10.sup.3                                           Culisetta sp.          3 * 10.sup.3                                           Culex (Neoculex) deserticola                                                                         2 * 10.sup.3 +                                         Culex tritaeniorhychus 4 * 10.sup.2                                           ______________________________________                                         +Concentration as a function of habitat was tested, this was the stronges     concentration employed for any habitat.                                  

                  TABLE III                                                       ______________________________________                                        Toxicity of ONR 60A Against Selected Species of                               Mosquito Larvae Showing % Mortality                                                      Concentration                                                      Species/Stage                                                                            (cells/ml)  Time Period                                                                              % Mortality                                 ______________________________________                                        Aedes sierrensis                                                                         10.sup.4    144 hours  45.5                                        First instar                                                                             10.sup.5    144 hours  92.2                                                   10.sup.6    144 hours  100.0                                       Aedes sierrensis                                                                         10.sup.4    140 hours  11.6                                        Third instar                                                                             10.sup.5    140 hours  95.0                                                   10.sup.6     17 hours  100.0                                       Culiseta incidens                                                                        10.sup.4    17.5 hours 0                                           Second instar                                                                            10.sup.5    17.5 hours 100.0                                                  10.sup.6    17.5 hours 100.0                                       Culiseta incidens                                                                        10.sup.4    17.5 hours 0                                           Third instar                                                                             10.sup.5    17.5 hours 100.0                                                  10.sup.6    17.5 hours 100.0                                       Culiseta incidens                                                                        10.sup.4    17.5 hours 0                                           Fourth instar                                                                            10.sup.5    17.5 hours 78.9                                                   10.sup.6    17.5 hours 100.0                                       ______________________________________                                    

Table IV illustrates the toxic effects of ONR-60A against selectednon-target organisms. Note that only the genus Dixa, closely related tomosquitoes was affected. The planktonic crustaceans tested showed nomortality due to the entomotoxin. As these groups of animals are thebase of the food pyramid in most aquatic ecosystems, the lack of adverseeffects is quite important. Further, in agricultural screening testsdevised from criteria established by the EPA, ONR-60A showed no usefullarvicidal activity against the target agricultural insects, thusclearly indicating a totally unique strain of Bacillus thuringiensis.Still further, ONR-60A demonstrated no significant loss in larvicidalactivity following heat shock for 20 minutes at 60° C. or followingexposure to ultraviolet radiation (2537 A°) sufficient to reduce theviable spore count to less than 0.1% of its initial value. Hence, toxicactivity can be attributed to an ultraviolet and heat-stable endotoxin.

                  TABLE IV                                                        ______________________________________                                        Toxicity of ONR-60A Against Selected                                          Non-Target Organisms                                                                          Concen-                                                                       tration   Time      %                                         Species         (cells/ml)                                                                              Period    Mortality                                 ______________________________________                                        Hyallela azteca 10.sup.5  10 days   0                                                         10.sup.6  10 days   0                                         Simocephalus vetulus                                                                          10.sup.5  10 days   2                                                         10.sup.6  10 days   3                                         Macrocyclops, sp.                                                                             10.sup.5  5 days    0                                                         10.sup.6  5 days    0                                         Dugesia dorotocephala                                                                         10.sup.5  10 days   0                                                         10.sup.6  10 days   0                                         Hyla regilla    10.sup.5  10 days   0                                                         10.sup.6  10 days   0                                         Alotanypus venusta                                                                            10.sup.5  8 days    0                                                         10.sup.6  8 days    60                                        Dixa, sp.       10.sup.5  17.5 hours                                                                              --                                                        10.sup.6  17.5 hours                                                                              100                                       Gambusia affinus holbrooki                                                                    10.sup.7  14 days   0                                         Gyrinidae       10.sup.7  14 days   0                                         Cladocelans     10.sup.7  7 days    0                                         Amphipoda       10.sup.7  4 days    0                                         Dytiscidae      10.sup.6  4 days    0                                         Notonecta glauca                                                                              10.sup.6  4 days    0                                         Hemiptera       10.sup.6  4 days    0                                         Corixidae       10.sup.7  3 days    0                                         Planaria        10.sup.7  3 days    0                                         Simuliidae      10.sup.6  3 days    0                                         Tabanidae       10.sup.7  24 hours  0                                         Ephemeroptera   10.sup.7  24 hours  0                                         ______________________________________                                    

Table V illustrates comparative larvicidal activities of ONR-60A andBacillus sphaericus (SSII-1), a current very important microbialentomotoxin. ONR-60A possesses considerably superior larvicidal activityover B. sphaericus (SSII-1), approximately a 30-fold increase for ED₅₀against Culex pipiens (complex). It also shows similarly high activityagainst Anopheles sergentii.

                  TABLE V                                                         ______________________________________                                        Comparative Dose Response of Microencapsulated Formulation                    of ONR-60A vs 36-Hr N2X Agar Test Culture of                                  Bacillus Sphaericus (SSII-1) Against                                          Anopheles Sergenti and Culex Pipiens (complex)                                          Concentration (Cells/ml)                                                        Bacillus                                                          Larvae Species                                                                            sphaericus ONR-60A    % Mortality                                 ______________________________________                                        Culex pipiens                                                                             3.5 * 10.sup.5                                                                           2.8 * 10.sup.4                                                                           25                                          (complex)   8.2 * 10.sup.5                                                                           3.6 * 10.sup.4                                                                           50                                                      1.8 * 10.sup.6                                                                           5.5 * 10.sup.4                                                                           75                                                      1 * 10.sup.7                                                                             2.0 * 10.sup.5                                                                           100                                         Anopheles sergentii                                                                       No data    3.1 * 10.sup.5                                                                           *25                                                     No data    5.0 * 10.sup.5                                                                           *50                                                     No data    8.0 * 10.sup.5                                                                           *75                                                     No data    4.6 * 10.sup.6                                                                           *90                                         ______________________________________                                         *Response time = 6 hours                                                 

It must be emphasized that in the screening of clonal isolates to obtainunique biological strains of spores possessing unusually high larvicidalactivity, two classes of agar surfaces should be employed. One classshould be the class of agars which elicit toxin production by currentbacterial organisms such as Bacillus sphaericus (SSII-1). An examplewould be the agar in commercial use for such production, N2X. Use ofsuch a test agar quickly eliminates from consideration all spores notdemonstrating toxic activity when grown on an agar surface from thisclass as potentially of no commercial utility. The other class should bethe class of nutrient agars which do not normally elicit toxinproduction by bacterial organisms normally found in the soil. Theability of spores to form entomotoxins when grown on such surfaces seemscorrelated with toxin stability, an extremely important toxincharacteristic. In the instant invention, none of the other screenedspores, including Bacillus sphaericus (SSII-1), demonstrated usefultoxic activity when grown on the nutrient control agar surface. OnlyONR-60A demonstrated toxic activity independent of the agar surface onwhich grown. Thus employment of the nutrient control agar aids inisolating unique strains having stable toxin.

The present larvicide is effectively applied not to the adult mosquitoesbut to the larvae. As such, it is necessary in the present invention forthe larvae to ingest the spores of ONR-60A. The carrier mechanism bywhich ONR-60A is dispersed will now be considered. The dried spores ofONR-60A are produced for dispersal with the carrier by conventionaltechniques familar to those skilled in the art.

An oleaginous, non-mineral-derived liquid of choice is put into solutionwith dioxane. A number of dried spores of ONR-60A is then added to thesolution to form a colloidal suspension having concentration suitablefor the desired application. Addition of water to the suspension resultsin the dioxane diffusing in the water and the oleaginous liquidnucleating about and microencapsulating the cells of ONR-60A to form abuoyant, colloidal suspension wherein the particle density of the cellplus the nucleated liquid is less than one. The water may be added tothe first suspension and the second suspension transported to thedispersal site or the first suspension may be injected into the watercausing the second suspension to then formulate at the dispersal site.

The oleaginous liquid to be employed must be hydrophilic in nature.Although the combination of spore and nucleated liquid about the sporepossesses positive buoyancy, the hydrophilic phenomena of the liquidinteracting with the surface tension phenomena of the water causes thesuspension to lie just below the surface of the water within theprinciple feeding zone of the mosquito larvae. Examples of, although byno means limited to, such liquids are corn oil, cottonseed oil andlanolin in combination with some conventional wetting agent well-knownto those in the art. The liquid preferred for formulating the buoyantcolloidal suspension is jojoba oil as field tests indicate thehydrophilic properties of the oil may result in requiring little or noadditional wetting agent.

The buoyant character of this formulation of ONR-60A provides for theconcentration of the toxic colloid in the primary feeding zone ofmosquito larvae, i.e., in a liquid layer just below the surface. Oneliter of microencapsulated formulation of 10¹⁰ spores/ml of ONR-60A isprojected to provide, for example, a lethal concentration to mosquitolarvae over an area of 1000 m².

Obviously, as mentioned supra, trivial mutations of the instantmicroorganism and many modifications of the instant carrier means arepossible in light of the above teachings. Such trivial mutations of themicroorganism and such modifications of the carrier are contemplated andare within the scope of the appended claims.

What is claimed is:
 1. The empirical method of isolating a purebiological strain of spores of a microorganism having unique,larvae-specific, high-larvicidal activity against mosquito-like larvaefrom sample mediums, said sample mediums obtained from areas withinknown mosquito-like larvae habitats, said areas having uncommonly lowmosquito-like larvae populations and a plurality of unknown bacterialmicroorganisms, comprising the steps of:(a) locating at least one zoneof uncommonly low larvae population within areas normally having highmosquito-like larvae populations; (b) taking soil samples from withinsaid at least one zone; (c) diluting said samples; (d) placing amountsof said diluted sample on a plurality of agar plates; (e) incubatingsaid diluted samples to obtain a first clearly-defined, visual clonalmorphology; (f) admixing sample spores of each of said first clonarmorphology with a sterile diluent to obtain a first test slurry; (g)challenging a first series of test wells with said first test slurry,each of said first test wells having a plurality of first testmosquito-like larvae; (h) retaining spores of a respective sample havingabout 80% mortality on said first test larvae within from about 24 toabout 48 hours; (i) inoculating said retained spores on both a controland a test agar surface; (j) incubating said spores on said control andsaid test agar surfaces to get a second and a third clearly defined,visual clonal morphology; (k) admixing sample spores of each of saidsecond and said third clonal morphology with a sterile diluent to obtaina second and a third test slurry; (l) challenging second and a thirdseries of test wells respectively with said second and said third testslurry, each of said test wells having a plurality of second testmosquito-like larvae; and (m) retaining spores of said second and saidthird test slurry each having about 80% toxicity on said second testlarvae, said spores of said second and said third test slurry havingtoxicity independent of the agar surface on which grown.
 2. The methodof claim 1 wherein the control agar surface is a nutrient agar.